The survival of developing vertebrate neurons is thought to be dependent on access to a limited supply of target-derived neurotrophic factors. Nerve growth factor (NGF) is the most extensively characterized of these factors and has been shown to regulate the survival of discrete populations of neurons.
U.S. Pat. No. 4,185,095 to Young discloses the isolation of a nerve growth factor from mouse submandibular or salivary gland.
U.S. Pat. No. 5,082,774 to Heinrich discloses a recombinant human nerve growth factor. The nerve growth factor is in an expression vector with an adenovirus strong, major, late promoter. Heinrich discloses that the expression vector may be used to stably transfect mammalian cells such as CHO or COS cells.
U.S. Pat. No. 5,180,820 to Barde et al. discloses that NGF has been molecularly cloned.
Various transgenic mice have been patented. U.S. Pat. No. 4,736,866 to Leder et al. discloses a transgenic non-human eukaryotic animal whose germ cells and somatic cells contain an activated oncogene sequence introduced into the animal or an ancestor of the animal at an embryonic stage. The embryo of the mice were microinjected with approximately 500 copies of the RSVS-107 c-myc plasmid. The injected eggs were transferred to pseudopregnant foster families and allowed to develop to term. The plasmid contains a Rous Sarcoma Virus enhancer and promoter sequence. The resultant mice showed expression of the c-myc gene in the salivary gland, spleen, testes, lung, brain, and preputial gland and intestinal tissue.
U.S. Pat. No. 5,175,383 to Leder et al. discloses a male transgenic mouse containing germ cells and somatic cells which contain a recombinant gene which is a vertebrate gene in the Int-2/FGF family which is capable of promoting benign prostatic hyperplasia or hypertrophy. The fusion gene which is injected into the mouse embryonic tissue comprises a promoter sequence controlling transcription of the recombinant gene such as a promoter derived from the mouse mammary tumor virus and cytomegalovirus. The recombinant gene is preferably substantially homologous with (i.e., greater than 50% homologous in terms of encoded amino acid sequence) a naturally occurring, vertebrate gene in the Int-2/FGF gene family of murine growth factor encoding genes or their vertebrate counterparts, including the murine acidic or basic fibroblast growth factor genes, the murine FGF-5 gene, the murine epidermal growth factor gene, the murine insulin-like growth factor-1 and -2 gene, the murine .alpha.-transforming growth factor gene with a murine hst/KS3 gene. The transgenic mice of Leder et al. exhibit prostate hyperplasia and give sterile offspring.
U.S. Pat. No. 5,175,384 to Krimpenfort et al. discloses transgenic mice having a phenotype characterized by the substantial absence of mature T-lymphocytes. The mouse is produced by introducing a transgene into a zygote of a mouse which comprises gene fragment which encodes a T-cell antigen receptor polypeptide variant which is incapable of mediating T-cell maturation in the transgenic mouse.
U.S. Pat. No. 5,175,385 to Wagner et al. discloses the production of a transgenic mouse with enhanced viral resistance which is transmissible to its offspring. The transgenic mouse is prepared by introduction of a gene encoding a human interferon having anti-viral activity into a host mouse. The plasmid of Wagner et al. contains a methallothionein-1 promoter and the genomic human beta interferon gene. When these mice were injected with pseudorabies virus they showed an increased resistance to the virus and although many mice died they died considerably later than did the control animals.
Vassar et al., in the Proceedings of the National Academy of Sciences, Volume 86, pages 1563-1567, March 1989, disclose "Tissue-specific and Differentiation-specific Expression of a Human K14 Keratin Gene in Transgenic Mice". This publication discloses that Vassar et al. used a plasmid containing a K14 keratin promoter and a neuropeptide substance P and determined the expression of the plasmid and neuropeptide substance P in the tail skin of transgenic mice.
Vassar et al. in Gene & Development, Volume 5, pages 714-727, (1991) disclose "Transgenic Mice Provide New Insights into the Role of TGF-.alpha. During Epidermal Development and Differentiation". This publication discloses the construction of transgenic mice using a plasmid which contains the K14 keratin promoter and the TGF-.alpha. gene. Vassar et al. disclose that the TGF-.alpha. gene belongs to the epidermal growth factor family of proteins and shows structural homology with the epidermal growth factor. These transgenic mice showed gross phenotypic abnormalities in their skin including flaky outer epidermal layers with stunted hair growth and wrinkling. The plasmid used to create this transgenic mouse also included human growth hormone fusion gene.
Patil et al. in Neuron, Volume 2, pages 347-447, March 1990, disclose "Specific Neuronal Expression of Human Nerve Growth Factor Receptors in the Basal Forebrain and Cerebellum of Transgenic Mice". Patil et al. employed a cosmid clone containing the entire NGF receptor gene. Introduction of the human NGF receptor gene into the mouse germ line resulted in significant levels of expression in specific cells in both the peripheral and central nervous systems. The NGF receptor gene and the NGF gene are distinct genetic material.
Borrelli et al. in Proceedings of the National Academy of Science, Volume 89, pages 2764-2768, April 1992, disclose "Pituitary Hyperplasia Induced by Ectopic Expression of Nerve Growth Factor". Borrelli et al. fused nerve growth factor cDNA to rat prolactin promoter to induce its ectopic expression in pituitary lactotrophs of transgenic mice.
Federoff et al. in Proceedings of the National Academy of Science, USA, Volume 89, pages 1636-1640, March 1992, disclose "Expression of Nerve Growth Factor in Vivo from a Defective Herpes Simplex Virus-1 Vector Prevents Effects of Axotomy on Sympathetic Ganglia". This publication teaches that rat nerve growth factor packaged into a herpes simplex virus-1 particle after axotomy of sympathetic superior cervical ganglion prevented a decline in the tyrosine hydroxylase level which is generally required for maintenance of the noradrenergic neurotransmitter system.
Edwards et al. in the Cell, Volume 58, pages 151-170, Jul. 14, 1989, disclose "Directed Expression of NGF to Pancreatic .beta. Cells in Transgenic Mice Leads to Selective Hyperinnervation of the Islets". Edwards et al. showed a selective increase the expression of NGF in pancreatic .beta. cells by creating a hybrid gene that contained a rat insulin promoter fused to mouse NGF cDNA followed by an intron and transcriptional terminator from the SV40 virus.
PCT Publication WO 93/00909 is directed to a method of treating neurotrophin-expressing tumors by administering a sequence complementary to at least a portion of a RNA transcript of brain-derived neurotrophic factor gene.
During development of the vertebrate nervous system up to half of all neurons generated undergo a process of naturally occurring cell death (See Ard, M. D., Morest, D. K. (1984). Cell death during development of the cochlear and vestibular ganglia of the chick. Intl. J. Dev. Neurosci. 2, 535-547; and Oppenheim, R. W. (1991). Cell death during development of the nervous system. Ann. Rev. Neurosci. 14, 453-501).
Neuronal death typically occurs shortly after neurons being making functional connections within their target field. Survival during this period of innervation is thought to be dependent on the synthesis by the target tissue of a limited quantity of neurotropic substances. (See Thoenen, H. and Barde, Y. A. (1980). Physiology of nerve growth factor. Physiol. Rev. 60, 1284-1334; Oppenheim, R. W. (1989). The neurotrophic theory and naturally occurring motoneuron death. Trends in Neurosci. 12, 252-255; and Davies, A. M., Larmet, Y., Wright, E., Vogel, K. S. (1991). Coordination of trophic interactions by separate developmental programs in sensory neurons and their target fields. J. Cell Sci. 15, 111-116)).
The competition for- target field-derived neurotrophic factors is though to serve at least two functions: to ensure that an appropriate number of synaptic contacts are made at the target and to eliminate inappropriate neuronal projections. (See Oppenheim, R. W. (1981). Neuronal death and some related phenomena during neurogenesis. In Studies in Developmental Biology (ed. W. M. Cowan) p 74-133. Oxford University Press; and Cowan, W. M., Fawcett, J. W., O'Leary, D.D.M., and Stanfield, B. B. (1984). Regressive events in neurogenesis. Science 225, 1258-1265)).
This concept is referred to as the neurotrophic hypothesis and predicts that the number of neurons in the adult could be increased if a higher concentration of neurotrophic factor(s) was available during the critical time when synaptic contacts are being established.
Nerve growth factor (NGF) is a prototypical target-derived neurotrophic substance that has been shown to be essential for the survival and differentiation of neural crest-derived sensory neurons, sympathetic neurons, and forebrain cholinergic neurons. (See Levi-Montalcini, R. and Booker B. (1960). Destruction of the sympathetic ganglia in mammals by an antiserum to a nerve-growth protein. Proc. Natl. Acad. Sci. U.S.A 46, 384-391 and Hefti, F. (1986) J. Neurosci. 6, 2155-2161; Williams, L. R., Varon, S., Peterson, G., Wictorin, K., Fischer, W., Bjorklund, A. & Gage, F. H. (1986). Proc. Natl. Aced. Sci. U.S.A. 83, 9231-9235).
The role of NGF in neuron survival is supported by numerous studies that have utilized both tissue culture and in vivo approaches in which NGF peptide or NGF antibodies were applied. (See Levi-Montalcini and Booker, 1960, supra; Johnson, E. M., Gorin, P. D., Brandeis, L. D., Pearson, J. (1980). Dorsal root ganglion neurons are destroyed by exposure in utero to maternal antibody to nerve growth factor. Science 210, 916-918; Barde, Y. A., Edgar, D., Thoenen H. (1980). Sensory neurons in culture: Changing requirements for survival factors during embryonic development. Proc. Natl. Acad. Sci. U.S.A. 77, (1980) 1199-1203; Hamburger, V. and Yip, H. W. (1984). Reduction of experimentally induced neuronal death in spinal ganglia of the chick embryo by nerve growth factor. J. Neurosci. 4, 767-774). Sympathetic and certain sensory neurons can be reduced by exogenous NGF whereas anti-NGF antibodies eliminate these neurons.
During development of skin, NGF is expressed by target cells of the presumptive epidermis and dermis. (See Davies, A. M., Bandtlow, D., Heumann, R., Korsching, S., Rohrer, H., and Thoenen, H. (1987). Timing and site of nerve growth factor synthesis in developing skin in relation to innervation and expression of the receptor. Nature 326, 353-358).
Target-field synthesis of NGF coincides with the arrival of axons to the epidermis, though the onset of expression as well as the concentration of NGF are independent of innervation. (See Rohrer, H., Heumann, R., Thoenen, H. (1988). Synthesis of nerve growth factor (NGF) in developing skin is independent of innervation. Dev. Biol. 128, 240-244; and Harper, S. J., and Davies, A. M. (1990). NGF mRNA expression in developing cutaneous epithelium related to innervation density. Development 110,515-519). Thus, cells of the target field appear to control the onset and level of NGF gene expression.
In mouse whisker pad skin, time course studies of innervation have shown that by embryonic day 11 (E11) axons have grown out to the skin and NGF mRNA expression has begun. (See Davies et al., 1987, supra). NGF continues to rise until E14 and then falls sharply by E15. The decrease in NGF concentration coincides with the onset of death of nearly half of the neurons in the trigeminal ganglion.
As skin innervation proceeds, the embryonic epithelium begins the transformation from a two-cell layer, undifferentiated periderm to a multilayer, stratified epidermis. (See Schweizer, J., and Winter, H. (1982). Keratin polypeptide analysis in fetal and in terminally differentiating newborn mouse epidermis. Differentiation 22, 19-24; and Kopan, R. and Fuchs, E. (1989). A new look into an old problem: Keratins as tools to investigate determination, morphogenesis, and differentiation in skin. Genes and Devel. 3, 1-15).
The differentiation of the epidermis is marked by the expression of epidermal specific genes, one of the earliest being the gene encoding the keratin intermediate filament protein K14. The onset of K14 gene expression in mouse skin occurs at approximately E14-E15 (see FIG. 1(b), beginning as NGF concentration is declining.
Heretofore there has been no convenient method to investigate how target-derived NGF affects neuron survival and development of the peripheral nervous system or provide a practical model for testing drug candidates for the treatment of neurodegenerative disorders of the brain such as Parkinson's syndrome and Alzheimer's disease.
The present invention overcomes the deficiency by providing transgenic mice that manufacture increased levels of NGF in the basal keratinocytes of the skin and exhibit hyperinnervation.